Project summary The Wnt pathway plays a crucial role in embryonic development and disease. During early development, Wnt signaling is required for gastrulation and mesendoderm differentiation, and, slightly later, for the differentiation of neural crest cells within the ectodermal layer. While the detailed biochemistry of signal transduction has been studied extensively, much less is known about the dynamics of pathway activity. We have used CRISPR-Cas9 genome engineering to create a novel reporter for Wnt pathway dynamics in both human embryonic stem cells (hESCs) and human cancer cells with temporal resolution on the time scale of minutes. Using this tool, we have found that stimulation by Wnt ligands yields transient localization of the signaling effector ?-catenin to the cell nucleus. This was surprising as previous work has shown, and we have confirmed with our reporter, that total ?-catenin levels are stably elevated by Wnt signaling. Here, we propose to use a combination of automated fluidic control for manipulating ligand dynamics, quantitative measurements with Wnt pathway and cell fate reporters, and mathematical modeling to dissect the relationship between ligand inputs, signal transduction by ?-catenin, and the resulting cell fate decisions. We will examine the role of endogenous Wnt signaling in human ectodermal patterning using a novel hESC culture platform. In this system, cells are grown in controlled geometries using micropatterning technology, differentiated to ectodermal fates, and then treated with the ligand BMP4. We have shown that BMP4 response leads to epidermal fates near the colony border, and that it induces secondary, endogenous Wnt signals that lead to neural crest differentiation at a particular radius within the colony. Cells at the center of colony do not receive these signals, and adopt a neural fate. We will combine this assay with live cell measurements of Wnt and BMP pathway activities and cell fate markers to understand how the interplay between the supplied BMP signal and the induced Wnt signal creates self-organized patterns of ectodermal cell fates. This project will provide essential information on Wnt pathway dynamics, which is crucial for understanding its role in embryonic development, harnessing it in directed differentiation protocols, and circumventing it in cases where Wnt signaling drives the growth of cancers. Further, understanding the role of Wnt in the in vitro ectodermal patterning assay will reveal principles of generating self-organized tissues in vitro, a central goal in regenerative medicine.